Historically maize (corn) has been used as a source of food for human and animal consumption. Even today maize supplies about twenty percent of the world's calories. Any maize disease that is prevalent in a large range of the maize growing regions can have a substantial impact on the quantity of maize annually available for consumption. Thus disease resistant maize is understandably of great public interest. And likewise is the subject of particular interest in many corn breeding programs.
To date disease resistant maize has been developed by traditional breeding methods; unfortunately, there is usually a trade off between desirable agronomic traits and disease resistant plants. In fact, it is quite common to see poor agronomic traits associated with moving disease or insect resistance traits (genes) especially when the resistance is moved from unadapted germplasm into adapted germplasm. Rarely can disease resistant maize having agronomically desirable traits such as high yield in the hybrid combination be achieved by traditional plant breeding. One example of a disease resistant plant that has not yet been developed by traditional breeding is a MCDV resistant corn.
There is a need for MCDV resistant corn because plant damage from MCDV is found in a number of corn growing regions. Certain sections of these regions are more prone to crop loss from MCDV than are other regions. In corn plants damage from MCDV may result in yield decrease, plant loss, or failure to develop ears.
MCDV is transmitted in nature by an insect. The insect is a leafhopper, Graminella nigrifrons (Forbes). MCDV occurs where the leafhopper, its vector, and the virus' over-wintering host johnsongrass (Sorghum halapense) are located. At the beginning of the growing season the leafhoppers acquire the virus from infected johnsongrass and transmit it to the corn as the insects move about the field. An infected corn plant is identifiable because the virus causes certain characteristic symptoms including, but not limited to, veinbanding, leaf twisting, leaf margin tearing, and chlorosis of the whorl.
Traditionally there have been two principle ways of controlling MCDV, one is the rotation with soybeans, and the second is johnsongrass management. There is a need for the development of a virus resistant plant effective to control MCDV. The biggest problem with developing MCDV resistance has been with disease escapes, which are a function of insect behavior and whether or not they are viruliferous.
Additionally, under field conditions it has been difficult to positively diagnose this particular virus disease based on symptom expression. Controlled greenhouse screens have been undertaken to: (1) insure infection of a plant with the virus; and (2) evaluate the response of maize to the infection.
A screening procedure for the detection of resistance to MCDV permits inbred lines to be evaluated under freedom of choice conditions by the vector but in the absence of Maize Dwarf Mosaic Virus (MDMV) (which can evidence similar symptoms). Graminella nigrifrons adult leafhoppers are used as vectors of MCDV. These vectors acquire the virus by exposure to infected corn and then are released in intervals into a cage containing the seedling corn plants. The original leafhoppers are not removed even though two newly acquired groups of leafhoppers are added to the cage at timed intervals. This (multiple inoculation method) effectively infects the corn seedlings so their MCDV response can be rated.
Hitherto, few, if any, agronomically desirable varieties of corn having resistance to MCDV and also the necessary agronomic traits for commercial production have been produced. Some moderately tolerant sources are known but the genetic background of these frequently evidence agronomically undesirable characteristics. Given that pursuant to this invention it has been discovered that a large number of genes control both mild symptomatic response to MCDV, a progeny plant containing the desired mix of agronomic traits and MCDV resistant genes within its genome is expected to be a very rare occurrence indeed.
One of the fundamental principles of maize breeding whether for disease resistance or otherwise, is the production of a hybrid having a desired mix of traits by the combination of two inbreds. To produce improved hybrids, there is an ongoing development of new inbreds. An inbred is a plant which has become homozygous at almost all loci. There are two primary germplasm sources for producing new inbreds. One source is germplasm that has been genetically engineered; the second source is an adapted or an unadapted germplasm. This invention relates to the use of unadapted germplasm and not to genetically engineered germplasm.
In a conventional breeding program, pedigree breeding and recurrent selection breeding methods are employed to develop new inbred lines with desired resistant traits. Maize breeding programs attempt to develop these inbred lines by self-pollinating plants and selecting the desirable plants from the populations. An inbred produces a uniform population of hybrid plants when crossed with a second homozygous line, i.e., inbred. Inbreds tend to have poor vigor and low yield; however, the progeny of an inbred cross usually evidences vigor. The progeny of a cross between two inbreds is often identified as an F.sub.1 hybrid. The resultant F.sub.1 hybrids which may be heterozygous at a number of loci, are evaluated to determine whether or not they show the resistant trait and agronomically important and desirable traits. Identification of desirable agronomic traits has typically been done by breeders' expertise. A plant breeder identifies a desired trait for the area in which his plants are to be grown and selects inbreds which appear to pass the desirable trait or traits on to the hybrid.
Conventional plant breeders rely on phenotypic traits of the inbreds for selection purposes. Modern plant breeding technology looks at the genotypic material (chromosomes) for plant breeding purposes. One method of looking at plant genotypes is to use Restriction Fragment Length Polymorphisms (RFLPs) which provide a method for identifying the chromosomal regions which affect the agronomic traits in the plant genome which the plant breeder is attempting to introgress into the inbred line for ultimate expression in the hybrid.
RFLPs can be used to identify chromosomal regions in maize which is a ten chromosome plant. Each chromosome has a short arm with a distal and proximal end and a long arm having a distal and proximal end. Between the short arm proximal end and long arm proximal end is a centromere. Each chromosome is made up of strands of the deoxyribonucleic acid (DNA) molecule which has a specific nucleic acid sequence. Selected restriction endonucleases will identify a specific base sequence and cleave the DNA molecule wherever this sequence occurs. The resultant cleaved portions are called restriction fragments. These restriction fragments can be separated by size by electrophoresis through agarose gels.
The DNA of two individual maize plants will differ in sequence at a variety of sites. Because of this difference, restriction endonucleases may cleave an individual's DNA at a different site or location than the other individual's DNA. A polymorphism in the length of restriction fragments is produced when the fragments of the two individuals have different lengths. A polymorphism is detected by placing the fragments on an agarose gel electrophoresis apparatus and allowing them to separate by size over distance. A southern blot is then used. The fragments of the DNA are physically transferred on to a membrane, then nucleic acid hybridization detects the sequences by hybridization of the single strand of DNA (probe) on the southern blot. The nucleic acid reforms double stranded DNA. A radiolabelled probe is used to detect a particular (DNA) sequence. One method is to use a labelled probe such that the DNA fragment will be identifiable through autoradiography techniques.
A variety of maize genes have been mapped and identified using RFLPs. Certain polymorphisms (molecular markers) are used to identify chromosomal areas associated with certain traits. A large number of molecular markers including RFLPs have been applied to the maize genome and a detailed maize genetic linkage map that can be used to localize important genes has been constructed.
A variety of traits have been identified by RFLPs; for example, P1 pericarp color has been linked to UMC185(P1) on the short arm of chromosome one of the maize plant. Probes BNL6.29 and UMC85 on chromosome six of the maize plant have been identified with Maize Dwarf Mosaic Virus (MDMV) strain A resistance in maize. Likewise, a variety of other traits have been genetically identified and placed on the maize genetic linkage map.
It would appear that once a desired trait is recognized and the chromosome region expressing that trait is located between flanking probes in a maize plant by the use of RFLPs, that the trait should be readily introgressed into an inbred line. Unfortunately, it is not easy to recognize the desired gene location and although RFLPs are a tool which can be employed to help identify the chromosomal region to which the trait appears to be linked, RFLPs are not a solution in and of themselves. RFLPs are simply a tool of identification. It should be noted that the chromosomal regions associated with MCD resistance have not been identified or mapped using probes.
Because of the difficulties with working with the MCDV trait, almost nothing has hitherto been known about the genes responsible, for example, the number of genes involved, their action, and where they are located on the maize chromosomes. There is a need for the identification of the location of genes associated with resistance to MCDV which permit their tracking when introgressed into new plants through traditional breeding. There also remains a need for a method of transferring resistance to MCDV to a corn inbred that has desirable agronomic traits. There remains a need for MCDV-resistant inbreds and hybrids.